Hydrocarbons, C9-C11, n-alkanes, isoalkanes,

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 January 2014 to 20 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

EXAMPLE:
- Concentrations:
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Water samples were taken from the control (replicates R1 – R2 pooled) and each loading rate WAF test group (replicates R1 - R2 pooled) at 0 and 48 hours (see Appendix 3).

- Sampling method:
Water samples were taken from the control (replicates R1 – R2 pooled) and each loading rate WAF test group (replicates R1 - R2 pooled) at 0 and 48 hours (see Appendix 3). Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

- Sample storage conditions before analysis:
Samples used immediately.Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
EXAMPLE

Vehicle:

no

Details on test solutions:

Range-finding Test
The loading rate to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.

Amounts of test item (11.25, 112.5 and 1125 mg) were each separately added to the surface of 11.25 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. The stirring vessels had minimal headspace and were sealed to prevent any potential losses due to test item volatility. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21 C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each
300 mL test and control vessel contained 300 mL of test media and was sealed to prevent any potential losses due to the volatile nature of the test item. After 24 and 48 hours the number of immobilized Daphnia magna were recorded.

The control group was maintained under identical conditions but not exposed to the test item.

A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.


Definitive Test
Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/L to confirm that no immobilization or adverse reactions to exposure were observed.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Appendix 1) in a temperature controlled room at approximately 19 deg C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

A positive control (Harlan Study Number 41301833) used potassium dichromate as the reference item. Details of the positive control are given in Appendix 2. The positive control was conducted between 16 May 2013 and 18 May 2013.


Test Water
Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined below.

Reconstituted Water – ISO Medium
Ingredient Final Concentration
(mg/L)
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 64.75
KCl 5.75

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

The results of the water quality measurements are given in Table 3. Temperature was maintained at 21 °C to 23 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The 0-Hour temperatures were measured to be slightly in excess of the 18 to 22 ºC (i.e. 23 °C) given in the study plan. This was considered not to affect the results of the test as no significant immobilization was observed and this temperature was not maintained for the duration of the

pH:

The results of the pH measurements are given in Table 3

Dissolved oxygen:

The results of the dissolved oxygen measurements are given in Table 3

Salinity:

freshwater used

Nominal and measured concentrations:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

Preliminary work (see Appendix 4) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.

No immobilization was observed at 1.0, 10 and 100 mg/L loading rate WAF.

Based on this information, a single loading rate of four replicates, of 100 mg/L, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.

Details on test conditions:

A nominal amount of test item (231 mg) was added to the surface of 2.31 liters of test water to give the 100 mg/L loading rate. The stirring vessel had minimal headspace and was sealed to prevent any potential losses due to test item volatility. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 100 mg/L loading rate WAF.

The concentration and stability of the test item in the 100 mg/L loading rate WAF test preparation was verified by chemical analysis at 0 and 48 hours (see Appendix 5).


Exposure Conditions
As in the range-finding test 300 mL test vessels containing 300 mL of test preparation were used. The vessels were sealed to prevent any potential losses due to the volatile nature of the test item. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were maintained in a temperature controlled room at approximately 21 °C to 23 °C with a photoperiod of 16 hours light (481 to 550 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period.


Evaluations
Test Organism Observations
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.


Water Quality Criteria
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.


Chemical Analysis of Test Loading Rates
Water samples were taken from the control and the 100 mg/L loading rate WAF test group at
0 hours and 48 hours (replicates R1 – R4 pooled) for quantitative analysis. Samples were stored frozen prior to analysis.

Duplicate samples were taken and stored frozen for further analysis if necessary.

The method of analysis, recovery and test preparation analyses are described in Appendix 5.


Statistical Analysis
An estimate of the EL50 values was given by inspection of the immobilization data.


Validation Criteria
The results of the test are considered valid if the following performance criteria are met:

• No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
• The dissolved oxygen concentration at the end of the test is ≥3 mg/L in the control and test vessels.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

RESULTS
Validation of Mixing Period
Preliminary investigational work (see Appendix 4) indicated that there was no increase in the amount of dissolved test item when the preparation period was extended for longer than
24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Range-finding Test
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.

No immobilization was observed at 1.0, 10 and 100 mg/L loading rate WAF.

Based on this information, a single loading rate of four replicates, of 100 mg/L, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.

Chemical analysis of the test preparations at 0 and 48 hours (see Appendix 5) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.98 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.


Definitive Test
Chemical Analysis of Test Loading Rates
Chemical analysis of the test preparations at 0 and 48 hours (see Appendix 5) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.98 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.


Immobilization Data
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.
There was no significant immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time (h) EL50
(mg/L Loading Rate WAF)
24 >100
48 >100

A single immobilized Daphnia was observed in the 100 mg/L loading rate WAF test group after 48 hours exposure. However, this was considered not to be a true toxic effect of the test item as this only represented 5% immobilization.

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Validation Criteria
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Water Quality Criteria
The results of the water quality measurements are given in Table 3. Temperature was maintained at 21deg C to 23 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The 0-Hour temperatures were measured to be slightly in excess of the 18 to 22 ºC (i.e. 23 °C) given in the study plan. This was considered not to affect the results of the test as no significant immobilization was observed and this temperature was not maintained for the duration of the test.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAF.

At the start of the mixing period the 100 mg/L loading rate was observed to be a clear colorless water column with an oily layer of test item floating on the surface. After 23 hours stirring and a 1-Hour standing period the 100 mg/L loading rate was observed to remain a clear colorless water column with an oily layer of test item floating on the surface. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 100 mg/L loading rate was observed to be a clear, colorless solution.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate were within the normal range for this reference item, see appendix 2.

Reported statistics and error estimates:

Statistical Analysis
An estimate of the EL50 values was given by inspection of the immobilization data.

Validity criteria fulfilled:

yes

Conclusions:

CONCLUSION
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Executive summary:

SUMMARY

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

Methods…….

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21°C to 23 °C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Results…….

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.98 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

 

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

 

Exposure ofDaphnia magnato the test item gave an EL₅₀value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

 

It was considered unnecessary and unrealistic to test at loading rates in excess of
100 mg/L loading rate WAF.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 January 2014 to 08 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Validation of Mixing Period
Preliminary work (see Appendix 4) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.


Range-finding Test
The loading rates to be used in the definitive test were determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.

Amounts of test item (22.5, 225 and 2250 mg) were each separately added to the surface of
22.5 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. The stirring vessels had minimal headspace and were sealed to prevent any potential losses due to test item volatility. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 21 C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. The test vessels were completely filled and sealed 300 mL stoppered conical flasks to prevent any potential losses due to test item volatility. After 24 and 48 hours the number of immobilized Daphnia magna were recorded.

The control group was maintained under identical conditions but not exposed to the test item.

A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.


Definitive Test
Based on the results of the range-finding test and after discussion with the Sponsor, the following loading rates were assigned to the definitive test: 10, 18, 32, 56, 100 and 180 mg/L.

Vehicle:

no

Details on test solutions:

Procedure
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item

Experimental Preparation
Amounts of test item (225, 405, 720, 1260, 2250 and 4050 mg) were each separately added to the surface of 22.5 liters of test water to give the 10, 18, 32, 56, 100 and 180 mg/L loading rates respectively. The stirring vessels were completely filled and sealed to prevent any potential losses due to test item volatility. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 10, 18, 32, 56, 100 and 180 mg/L loading rate WAFs.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours (see Appendix 5). The 0-Hour samples were stored frozen prior to analysis with the 48-Hour samples.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium
(see Appendix 1) in a temperature controlled room at approximately 19 C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

A positive control (Harlan Study Number 41301833) used potassium dichromate as the reference item. Details of the positive control are given in Appendix 2. The positive control was conducted between 16 May 2013 and 18 May 2013.


Test Water
Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined in Appendix 3.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

The results of the water quality measurements are given in Table 3. Temperature was maintained at approximately 21 deg C throughout the test.

pH:

The results of the water quality measurements are given in Table 3.

Dissolved oxygen:

The results of the water quality measurements are given in Table 3.

Salinity:

freshwater used.

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.

Based on the results of the range-finding test and after discussion with the Sponsor, the following loading rates were assigned to the definitive test: 10, 18, 32, 56, 100 and 180 mg/L.

Details on test conditions:

As in the range-finding test the 300 mL test vessels were completely filled and sealed to prevent any potential losses due to test item volatility. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then maintained in a temperature controlled room at approximately 21 deg C with a photoperiod of 16 hours light (444 to 481 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period.


Evaluations
Test Organism Observations
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.


Water Quality Criteria
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.


Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

RESULTS
Validation of Mixing Period
Preliminary investigational work (see Appendix 4) indicated that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.


Range-finding Test
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.

No immobilization was observed at 1.0 and 10 mg/L loading rate WAF. However, 30% immobilization was observed at 100 mg/L loading rate WAF.

Based on this information and after discussion with the Sponsor, loading rates of 10, 18, 32, 56, 100 and 180 mg/L were selected for the definitive test.

Chemical analysis of the 10 and 100 mg/L loading rate WAF test preparations at 0 and 48 hours (see Appendix 5) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.19 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.


Definitive Test
Chemical Analysis of Test Loading Rates
Chemical analysis of the test preparations at 0 and 48 hours (see Appendix 5) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.19 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.



Immobilization Data
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.

There was no immobilization in 20 daphnids exposed to a maximum nominal loading rate of 180 mg/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time (h) EL50
(mg/L Loading Rate WAF)
24 >180
48 >180

The No Observed Effect Loading rate after 24 and 48 hours exposure was 180 mg/L loading rate WAF.

A single Daphnid was observed immobilized at 48 hours in the 32 mg/L loading rate WAF test group. However, as this did not follow a concentration-dependent pattern and as no further immobilization was observed, this was considered not to be significant.

It was noted that a slight difference in immobilization was observed between the range-finding and definitive tests. However, it was considered that as a loading rate of greater than 100 mg/L was tested in the definitive test without immobilization being observed, the immobilization observed in the range-finding test at 100 mg/L loading rate WAF was considered not to be significant.


Validation Criteria
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.


Water Quality Criteria
The results of the water quality measurements are given in Table 3. Temperature was maintained at approximately 21 deg C throughout the test, while there were no treatment related differences for oxygen concentration or pH.


Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.


Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.

At the start of the mixing period the 10, 18, 32, 56, 100 and 180 mg/L loading rates were observed to be clear colorless water columns with an oily layer of test item floating on the surface. After 23 hours stirring and a 1-Hour standing period the 10, 18, 32, 56, 100 and
180 mg/L loading rates were observed to remain clear colorless water columns with an oily layer of test item floating on the surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 10, 18, 32, 56, 100 and 180 mg/L loading rates were observed to be clear, colorless solutions.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

An estimate of the EL50 values was given by inspection of the immobilization data.

Validation Criteria

The results of the test are considered valid if the following performance criteria are met:

 

·        No more than 10% of the controldaphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).

·        The dissolved oxygen concentration at the end of the test is ≥3 mg/L in the control and test vessels.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 180 mg/L loading rate WAF. The No Observed Effect Loading rate was 180 mg/L loading rate WAF.

This study showed that there were no toxic effects at the maximum concentration tested in the definitive test.

Executive summary:

SUMMARY

Introduction

A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 

 

Methods…….

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 18, 32, 56, 100 and 180 mg/L for 48 hours at a temperature of approximately 21°C under static test conditions. The number of immobilized Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

 

Results…….

Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.19 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

 

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

 

Exposure ofDaphnia magnato the test item gave EL₅₀values of greater than 180 mg/L loading rate WAF. The No Observed Effect Loading Rate was 180 mg/L loading rate WAF.

 

This study showed that there were no toxic effects at the maximum concentration tested in the definitive test.

Description of key information

No short-term aquatic invertebrate data exist for the registered substance. Reliable data do exist for related Fischer-Tropsch process-derived products in the relevant carbon number range.

The acute toxicity of the test items to the freshwater invertebrate Daphnia magna has been investigated and gave 48-Hour EL50 values of greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading rates were 100 mg/L loading rate WAF.

These data are read across to Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics. The results of the test are considered reliable.

Additional information

No measured invertebrate toxicity data are available for the registration substance itself. However, reliable data are available for related Fischer-Tropsch process-derived products in the relevant carbon number range. These are read across to Hydrocarbons, C9-C11, n-alkanes, isoalkanes, <2% aromatics.

 

Measured acute (short-term) toxicity data are available for Shell GTL Solvent GS160 (Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics) to the freshwater invertebrate Daphnia magna (Parr, 2014a). The test was conducted under static (no renewal of the test media) conditions in accordance with OECD Test Guideline 202 and method C2 of EC Regulation No. EC 440/2008. Appropriate modifications to the test and media preparation procedures were made to take account of the test substance containing multiple constituents, having low solubility in water and being potentially volatile. No effects on mobility of D. magna were observed after 48 hours exposure to the test medium prepared as a water-accommodated fraction (WAF) at a loading rate of 100 mg/l; 48-hour EL₅₀>100 mg/l, NOELR ≥100 mg/l. The results of the test are considered to be reliable.

Measured acute (short-term) toxicity data are available for Shell GTL Solvent GS170 (Hydrocarbons, C9-C12, n-alkanes, isoalkanes, <2% aromatics) to the freshwater invertebrate Daphnia magna (Parr, 2014b). The test was conducted under static (no renewal of the test media) conditions in accordance with OECD Test Guideline 202 and method C2 of EC Regulation No. EC 440/2008. Appropriate modifications to the test and media preparation procedures were made to take account of the test substance containing multiple constituents, having low solubility in water and being potentially volatile. No effects on mobility of D. magna were observed after 48 hours exposure to the test medium prepared as a water-accommodated fraction (WAF) at a loading rate of 180 mg/l; 48-hour EL₅₀>180 mg/l, NOELR ≥180 mg/l. Analysis of the 180 mg/l WAF at the start and end of the test showed it to contain less than 0.19 mg/l of dissolved constituents of the substance, which was the limit of quantification (LOQ) of the analytical method (GC-FID). The results of the test are considered to be reliable.

The water accommodated fraction (WAF) of hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics, did not produce a 50% effect (immobility) with Daphnia magna at a loading of 1000 mg/L after a 48-hour exposure. Therefore, the 48-hour LL₅₀ is reported as >1000 mg/L. There was no mortality at the 1000 mg/L loading level after 48 hours. Therefore, the 48-hour LL₀ for mortality is reported as 1000 mg/L. There was also no mortality in the control.

Water accommodated fractions (WAFs) of hydrocarbons, C9-C11, n-alkanes, isoalkanes, cyclics, <2% aromatics, did not produce a 50% effect (mortality) with Chaetogammarus marinus at loadings up to 1000 mg/L after a 96-hour exposure. Therefore, the 96-hour LL₅₀ is reported as >1000 mg/L. Mortality was observed in the 3 highest loadings, 100, 320, and 1000 mg/L after 96 hours, but no greater than 40% mortality was demonstrated and the two highest loading levels demonstrated only 20% mortality. Therefore, the 96-hour NOELR for mortality is reported as 32 mg/L. There was also no mortality in the control.